Abstract

Rationale: Hyperamylinemia is common in patients with obesity and insulin resistance, coincides with hyperinsulinemia, and results in amyloid deposition. Amylin amyloids are generally considered a pancreatic disorder in type 2 diabetes. However, elevated circulating levels of amylin may also lead to amylin accumulation and proteotoxicity in peripheral organs, including the heart.

Objective: To test whether amylin accumulates in the heart of obese and type 2 diabetic patients and to uncover the effects of amylin accumulation on cardiac morphology and function.

Methods and Results: We compared amylin deposition in failing and nonfailing hearts from lean, obese, and type 2 diabetic humans using immunohistochemistry and Western blots. We found significant accumulation of large amylin oligomers, fibrils, and plaques in failing hearts from obese and diabetic patients but not in normal hearts and failing hearts from lean, nondiabetic humans. Small amylin oligomers were even elevated in nonfailing hearts from overweight/obese patients, suggesting an early state of accumulation. Using a rat model of hyperamylinemia transgenic for human amylin, we observed that amylin oligomers attach to the sarcolemma, leading to myocyte Ca2+ dysregulation, pathological myocyte remodeling, and diastolic dysfunction, starting from prediabetes. In contrast, prediabetic rats expressing the same level of wild-type rat amylin, a nonamyloidogenic isoform, exhibited normal heart structure and function.

Conclusions: Hyperamylinemia promotes amylin deposition in the heart, causing alterations of cardiac myocyte structure and function. We propose that detection and disruption of cardiac amylin buildup may be both a predictor of heart dysfunction and a novel therapeutic strategy in diabetic cardiomyopathy.

Introduction

One-third of adults and 17% of children in the United States (from the National Center for Health Statistics, 2009) are currently obese and at high risk of developing both type 2 diabetes and cardiovascular disease.1–3 Progression to overt type 2 diabetes may accelerate pathological changes in heart structure and function,4–7 independent of confounding factors such as coronary artery disease and hypertension.1–3 It is assumed1 that increases in body fat can affect the body's response to insulin, potentially leading to insulin resistance and subsequent impaired glucose and lipid homeostasis. As such, insulin resistance is unequivocally associated with heart disease.1–10 However, the myocardial insulin responsiveness in diabetic patients is surprisingly intact,11,12 suggesting that factors secondary to insulin resistance may critically contribute to cardiac dysfunction in type 2 diabetes.5,9,10 In addition to hyperglycemia and dyslipidemia, patients with obesity and insulin resistance present also hyperinsulinemia and hyperamylinemia.13–15 Whereas the hyperinsulinemic response prevents a large fraction of insulin resistant patients from developing type 2 diabetes,1 the coincident hyperamylinemia leads to proteotoxicity and amyloid deposition.13,14 More than 95% of patients with type 2 diabetes stain positive for amylin amyloids in pancreatic islets.14 Amylin deposition was also found in kidneys of obese and type 2 diabetic patients.16 Recently,17,18 we hypothesized that hyperamylinemia may favor cardiac amylin accumulation,17 causing alterations of myocyte structure and function in ways that may contribute to progressive heart failure.18

Amylin is a 4-kDa hormone coexpressed and cosecreted with insulin by pancreatic β-cells.13,14 Human amylin, also known as islet amyloid polypeptide (IAPP), has aggregation properties similar to prions and amyloidogenic proteins that are associated with neurodegenerative diseases.19 At high secretion rates, amyloidogenic proteins readily form oligomers, fibrils, and amyloid plaques. It is increasingly recognized that soluble oligomers rather than fibrils and plaques are the most toxic species of amyloids.20–28 They attach to cellular membranes causing Ca2+ dyshomeostasis, cell dysfunction, and apoptosis.20–28 Previous data27,28 indicated that cardiac myocyte–restricted expression and accumulation of amyloidogenic peptides, such as polyglutamine27 or presenilin,28 can induce cytotoxicity and heart failure in mice. Presenilin oligomers coimmunoprecipitated with sarcoplasmic reticulum Ca-ATPase (SERCA) and altered Ca2+ handling in cardiac myocytes.28

To clarify whether amylin builds up in the heart and whether this could be associated with cardiac failure in obesity and type 2 diabetes, we assessed amylin deposition in hearts from lean, obese, and type 2 diabetic humans, with and without heart failure. Using a “humanized” rat model of hyperamylinemia, we examined changes in cardiac structure and function in relation with cardiac amylin accumulation.

Methods

Detailed procedures, description of human tissue specimens and animal models are included in the online-only Data Supplement.

Human Tissue Specimens

Failing hearts from obese, type 2 diabetic, and nondiabetic patients were obtained at the time of orthotopic heart transplantation at the Hospital of University of Pennsylvania. Nonfailing hearts from obese and lean individuals are from organ donation. Tissue specimens were obtained in accordance with institutional review board approval. Inclusion in tissue-based studies was not restricted on the basis of age, sex, race, or ethnic status. Heart failure etiology, body mass index, age, sex, and state of diabetes with respect to dependence on insulin and/or oral hypoglycemic agents of all cases studied here are summarized in Online Table I.

Experimental Animals

Studies were approved by the University of California, Davis, Animal Research Committee. Because rodent amylin is not amyloidogenic and rodents do not accumulate amylin amyloids,29 most rodent models of type 2 diabetes are not adequate for this study. We used Sprague-Dawley (SD) rats transgenic for human amylin in the pancreatic β-cells (HIP rats).30 HIP rat breeding pairs were kindly provided by Pfizer. HIP rats show hyperamylinemia, leading to amylin deposits in pancreatic islets and gradual decline in β-cell mass.31 They develop insulin resistance at 5 months of age and diabetes by 10 months of age.31 As negative controls, we used obese insulin-resistant rats expressing only wild-type, nonamyloidogenic rat amylin, which does not form amyloids (UCD-T2DM rats).32 UCD-T2DM rats were obtained by breading obese SD rats with Zucker Diabetic Lean rats that lack the leptin receptor defect and have inherent ß-cell defects.32 UCD-T2DM rats exhibit insulin resistance before the onset of diabetes,32 similar to HIP rats30 and humans.1 In the present study, we used age-matched HIP (n=17) and UCD-T2DM (n=19) rats in the prediabetic state, that is, nonfasting blood glucose level in the 150 to 200 mg/dL range.33 Wild-type littermates (n=16) served as nondiabetic controls for HIP rats. Age-matched SD rats (n=13, Charles Rivers Laboratory) were controls for UCD-T2DM rats.

Immunochemistry

Western blot analysis was performed on left ventricular homogenates, myocyte lysates, and blood serum. Immunohistochemistry was done on thin sections from paraffin blocks.

Electron Microscopy

Statistical Analysis

Data are expressed as mean±SEM. Statistical discriminations were performed using 2-tailed unpaired Student t test, with P<0.05 considered significant. One-way ANOVA with the Dunnett post hoc test was used when comparing multiple groups.

Results

We examined left ventricular tissue from 53 human hearts divided in 5 pathologically distinct groups (Online Table I). These included failing hearts from patients with type 2 diabetes (n=25) and obese patients that developed overt type 2 diabetes within 1 year after transplantation (n=8). These hearts were expected to show significant amylin accumulation. To uncover the early stage of amylin buildup in the heart, a third group included nonfailing hearts from overweight/obese humans (n=8). Last, nonfailing hearts from lean, healthy patients (n=5) and failing hearts from lean patients without diabetes (n=7), which should not accumulate amylin, served as negative controls.

To assess the level and size distribution of cardiac amylin aggregates, we performed Western blots with an anti-amylin antibody on left ventricular protein homogenates. We found molecular weight bands corresponding to amylin trimers (12 kDa), tetramers (16 kDa), and 2 additional larger molecular weight structures at ≈32 kDa (octamers) and ≈64 kDa (16-mers) (Figure 1A through 1C). Negative controls showed that these bands are specific (Online Figure I). Intensity signal analysis (Figure 1D through 1F) indicated that amylin oligomer accumulation is markedly larger in failing hearts from patients with type 2 diabetes and overweight/obesity than in normal hearts and failing hearts from patients without diabetes (controls). Intriguingly, large amylin oligomers, (32≥kDa) are abundant in failing hearts from diabetic and obese patients (Figure 1A, 1B, and 1F) but not in nonfailing hearts from overweight/obese individuals (Figure 1C and1F). Smaller amylin oligomers were already elevated in nonfailing hearts from overweight/obese patients (Figure 1C through 1E), indicating an early stage of cardiac amylin accumulation. The results are consistent with the idea that accumulation of large amylin oligomers can induce deleterious cardiac effects. Amylin tetramers are also present in failing hearts from nondiabetic patients (Figure 1E), which might indicate undiagnosed insulin resistance in those patients (as commonly seen in aging).

Immunohistochemistry with an anti-amylin antibody shows large amylin deposits in failing hearts from type 2 diabetic patients (Figure 2A through 2D), similar to those found in the pancreas of type 2 diabetic patients (Figure 2F). Amylin plaques (Figure 2A, 2C, and 2D) and fibrillar tangles (Figure 2B) are scattered through the entire heart. Amylin deposits are often seen at sites with myocyte multinucleation, variation in nuclear size and infiltrating cells, which usually occur with fibrotic and infiltrative diseases. In contrast, sections from normal hearts (Figure 2E) do not show amylin deposition and structural abnormalities. To quantify the extent of amylin deposition in large plaques and fibrils, pellets from heart protein homogenates were treated with formic acid and guanidine hydrochloride to partially break down the amylin oligomers. Dot blots showed significantly increased amylin levels (Online Figure II), indicating that fragmenting of large amylin aggregates enhanced detection by the anti-amylin antibody. This also implies that blots exhibiting higher order oligomers (Figure 1) probably underestimate the amount of amylin in these aggregates.

Amylin deposition in failing diabetic hearts demonstrated by immunohistochemistry with an anti-amylin antibody on thin heart sections. Amylin plaques (A, C, and D, arrows) and tangles (B, arrow) are scattered through the entire heart. E, Left ventricle section from a nonfailing heart; no amylin deposits are revealed. F, Positive control for amylin deposition in a pancreas from a diabetic patient.

Cardiac Amylin Accumulation Alters Ca2+ Cycling in HIP Rats

To test whether in vivo cardiac accumulation of human amylin affects Ca2+ cycling, we used prediabetic HIP rats. Age-matched, prediabetic UCD-T2DM rats expressing only the native, nonamyloidogenic rat amylin were used as negative control. Using prediabetic rats has the advantage that one can dissociate the effect of cardiac amylin accumulation from other confounding factors that affect cardiac Ca2+ cycling during late diabetes.7,38,39 Immunohistochemistry (Figure 4A) and dot blots (Figure 4B) with an anti-amylin antibody that recognizes both human and rat amylin (the latter with higher avidity) show that amylin significantly accumulates only in HIP rat hearts. Western blots on left ventricular homogenates and cardiac myocyte lysates from HIP rats (Figure 4C) show amylin multimers similar to those detected in humans (Figure 1A through 1C) in all groups. These data indicate that amylin oligomer accumulation in HIP rat hearts starts from prediabetes. The presence of amylin oligomers in cardiac myocyte lysates suggests that they attach to sarcolemma and/or enter the myocytes.

A, Immunohistochemistry with an anti-amylin antibody on thin heart sections demonstrating amylin deposition in cardiac tissues from prediabetic HIP but not UCD-T2DM rats (×20). B, Dot blots with the anti-amylin antibody comparing total amylin level in HIP versus UCD-T2DM rats. The first 2 dots on the left show positive controls using 5 ng of recombinant human (h-Amylin) and rat (r-Amylin) amylin. The antibody binds r-Amylin with significantly higher affinity than h-amylin; Bottom panel shows the average signal intensity in hearts from prediabetic HIP versus UCD-T2DM rats. The experiment was performed in triplicate. C, Representative Western blot with anti-amylin primary antibody on ventricular myocyte lysates from prediabetic HIP rats, and left ventricle protein homogenates from prediabetic (PD) and diabetic (DM) HIP rats. D, Blood glucose levels in age-matched prediabetic HIP rats (n=14) and UCD-T2DM rats (n=16). E and F, Akt phosphorylation in hearts from prediabetic HIP and UCD-T2DM rats and littermate controls under basal conditions (0 insulin) and after stimulation with insulin (10 mU/g body weight). Representative example (E) and mean values for the ratio between phosphorylated and total Akt (F); n=3 rats for each group.

To test whether cardiac amylin accumulation affects myocardial insulin responsiveness, we compared the activation status of Akt and GSK-3β, key components of the cardiac insulin signaling pathway, in HIP, littermate control, and UCD-T2DM rats. For this test, HIP and UCD-T2DM rats were matched for age and nonfasting blood glucose level (Figure 4D). The ratio between basal levels of phosphorylated Akt and total Akt is not statistically different among the three groups (Figure 4E and4F). Insulin stimulation significantly increased the phosphorylation of Akt in all rats and no statistical difference was observed among HIP, UCD-T2DM, and control groups (Figure 4E and4F). GSK-3β displays a similar response to stimulation by insulin (Online Figure VI).

Cardiac amylin accumulation in prediabetic HIP rats alters myocyte Ca2+ cycling (Figure 5). At low stimulation frequencies, Ca2+ transient amplitude is significantly larger (4.7±0.5 versus 3.5±0.3 at 0.5 Hz) in myocytes from prediabetic HIP rats versus control rats (Figure 5A and5D). In contrast, cardiac myocytes from age-matched, prediabetic UCD-T2DM rats show no change in Ca2+ transient amplitude (Figure 5G and Online Figure VII). Thus, cardiac amylin accumulation may be the cause for the larger Ca2+ transient amplitude in prediabetic HIP rats, in agreement with our results using exogenous human amylin oligomers (Figure 3). Different from littermate controls, the amplitude of Ca2+ transients in myocytes from prediabetic HIP rats decreases with increasing the stimulation frequency (negative staircase), so that at 2 Hz the amplitude is similar to that recorded in control rats (Figure 5B and5D). This is probably due to deficiencies in Ca2+ reuptake into the SR. Indeed, Ca2+ transient decline, which is mostly due to SR Ca2+ reuptake via the SR Ca-ATPase (SERCA), is significantly slower in prediabetic HIP rats versus control (τ=0.71±0.07 versus 0.55±0.04 s at a stimulation rate of 0.5 Hz;Figure 5C and5E). In contrast, Ca2+ transient decay remains unchanged in myocytes from age-matched, prediabetic UCD-T2DM rats (Figure 5H). Despite slower Ca2+ transient relaxation, the SR Ca2+ load, assessed as the amplitude of Ca2+ transient produced by 10 mmol/L caffeine, is similar in myocytes from control and prediabetic HIP rats paced at 2 Hz (ΔF/F0=8.5±0.4 versus 8.6±0.5). However, the slower Ca2+ transient relaxation in prediabetic HIP rats results in elevated diastolic [Ca2+]i at higher pacing rates (Figure 5B and5F). Diastolic [Ca2+]i is unaltered in prediabetic UCD-T2DM rats (Figure 5I). Similar to myocytes incubated with human amylin, the sarcolemmal Ca2+ leak was significantly larger in prediabetic HIP rats versus control (41.6±4.5 versus 29.2±2.4 104 · ΔF340/F380 · s−1, P<0.05). We infer that amylin oligomers can acutely increase Ca2+ leak into myocytes, causing elevated diastolic [Ca2+]i and Ca2+ transients, but that reduced SERCA function may be a longer-term effect, as in heart failure.

NFATc4 was also translocated to the nucleus in control rat myocytes incubated with 50 μmol/L human amylin for 2 hours (Online Figure VIII). At this concentration, human amylin forms oligomers and fibrils and elevates Ca2+ transients, as discussed in above. The distribution of HDAC4 was not altered by this acute amylin exposure (Online Figure VIII). We conclude that Ca2+-dependent nuclear signaling initiated by amylin oligomers is capable of inducing hypertrophic transcriptional effects.

Elevated natriuretic peptide levels are thought to reflect cardiac dysfunction and have been used as a “biomarker” of cardiac hypertrophy.42,43 We found that the level of brain natriuretic peptide (BNP) is elevated (by 100±30%) in hearts from prediabetic HIP rats versus littermate controls and further increases with diabetes development (Figure 6E). This suggests hormonal alterations specific to the onset of cardiac hypertrophy in HIP rats. In contrast, the BNP level is not altered in prediabetic UCD-T2DM rats and only increases after the full development of diabetes (Online Figure IX, A). Of note, a previous study found that external human amylin induces hypertrophy in isolated cardiac myocytes.44 However, the heart weight/body weight ratio in prediabetic HIP rats (2.72±0.22 g) versus control rats (2.71±0.2 g) did not change, showing the lack of overt cardiac hypertrophy in this early disease state.

Activation of Ca2+-dependent transcriptional pathways may also alter the transcription of key Ca2+ transport and regulatory proteins, which cause further alterations in Ca2+ cycling. Thus, we measured the protein expression of SERCA, phospholamban (the endogenous SERCA inhibitor), and Na+/Ca2+ exchanger, the main Ca2+ extrusion pathway in HIP rats (Figure 6F). We found that SERCA expression is reduced by 20% and 30% in prediabetic and diabetic HIP rats, respectively (Figure 6F). In contrast, SERCA expression was unchanged in prediabetic UCD-T2DM rats (Online Figure IX, B). Protein expressions of phospholamban and Na+/Ca2+ exchanger are unaltered in prediabetic HIP rats (Figure 6F).

Prediabetic HIP Rats Show Diastolic Dysfunction

To determine how amylin accumulation affects cardiac function, we performed in vivo echocardiography and hemodynamic measurements on prediabetic HIP rats (Table). We found significantly slower relaxation (reduced −dP/dtmin values) in prediabetic HIP rats versus control. This suggests that cardiac amylin oligomer accumulation may accelerate the occurrence of heart dysfunction, particularly diastolic dysfunction, a typical sign of diabetic cardiomyopathy.2–10,38,39 Furthermore, the left ventricular end diastolic volume is increased in prediabetic HIP rats, which, combined with the unchanged fractional shortening, suggests dilation of the heart (Table).

Discussion

We found significant accumulation of large amylin oligomers (>octamers,Figure 1A, 1B, and 1F), fibrillar tangles (Figure 2B), and plaques (Figure 2A, 2C, and 2D) in failing hearts from patients with obesity and type 2 diabetes, but not in normal hearts and failing hearts from lean humans without diabetes (Figure 1C and1F). Small amylin aggregates are even elevated in nonfailing hearts from overweight/ obese patients (Figure 1C through 1E), suggesting that cardiac amylin buildup starts in the early state of insulin resistance/prediabetes. HIP rats, which express human amylin in the pancreas, accumulate amylin oligomers in the heart (Figure 4A through 4C) starting also in prediabetes. In prediabetic HIP rats, the interaction of amylin oligomers with cardiac myocytes results in larger sarcolemmal Ca2+ leak and Ca2+ transients (Figure 5A and5D) leading to activation of Ca2+-mediated hypertrophic pathways (Figure 6A through 6D), pathological heart remodeling (Figure 6E and6F), and diastolic dysfunction (Figure 5E and5F and the Table). In contrast, UCD-T2DM rats, which are matched for age, blood glucose level (Figure 4D), and myocardial insulin responsiveness (Figure 4E and4F) but lack amylin deposition (Figure 4A and4B), have normal cardiac structure (Figure 4A and4B) and function (Figure 5G through 5I). These results suggest that cardiac dysfunction in HIP rats is most likely an amylin-mediated effect. Hence, hyperamylinemia and consequent amylin deposition, a toxic effect generally assumed to contribute to pancreatic β-cell dysfunction and development of type 2 diabetes,13,14,31,45,46 may also be causally implicated in cardiac dysfunction.

Pathologically Important Form of Amylin

Conditions underlying amylin oligomerization13–15,45,46 and proteotoxicity21,22 are complex and only poorly understood. Amyloidogenicity of human amylin promotes the attachment to the sarcolemma (Online Figure V, B) and oligomer formation, 2 apparently independent processes. Small oligomers develop rapidly at the sarcolemma, for example, in only 1–2 hours (Online Figure V), which correlates with an increase in sarcolemmal permeability to Ca2+ (Figure 3D and3E) and elevated Ca2+ transients (Figure 3A and3C), with consequent activation of Ca2+-mediated hypertrophic pathways (Online Figure VIII). In contrast, rat amylin, a nonamyloidogenic isoform of amylin29 (Online Figure III), does not affect Ca2+ cycling when incubated with cardiac myocytes (Figure 3B). These results suggest that the oligomers may be the pathologically important forms of amylin. Amylin oligomerization at the sarcolemma may act as seeds for further amyloid growth. Fibril growth at the membrane amplifies structural alteration of the membrane22 and Ca2+ dysregulation, aggravating the deleterious effects in the heart. This might be the case for the large amylin oligomers in the 32- to 64-kDa size range that are abundant in failing hearts from diabetic and obese patients (Figure 1A, 1B, and 1F) and in HIP rat hearts (Figure 4C).

There is increasing support for the toxic oligomer hypothesis in amyloid-related diseases,20–28 including cardiomyopathies caused by other amyloidogenic proteins that infiltrate the heart, for example, transthyretin, immunoglobulin light chain, and serum amyloid.47 Data48,49 suggest that the infiltration of amyloidogenic proteins in the heart may induce cardiotoxicity even before amyloid fibril formation. Moreover, intracellular accumulation of amyloid oligomers, such as those formed by polyglutamine27 or presenilin,28 induced cytotoxicity and heart failure in mice. Presenilin oligomers coimmunoprecipitated with SERCA and altered Ca2+ handling.28 Our data from human and HIP rat hearts suggest that amylin oligomer accumulation in the heart, which is an outside-inside cardiac event, is cardiotoxic and may represent an early pathogenic mechanism linking type 2 diabetes with cardiac dysfunction.

Cardiac Amylin Accumulation and Altered Myocyte Ca2+ Cycling

Our data show that the primary effect of cardiac amylin oligomer accumulation is an increase in myocyte Ca2+ and Ca2+ transients (schematic inFigure 7). This effect was observed both in cardiac myocytes exposed acutely to human amylin and in prediabetic HIP rats, which accumulate amylin oligomers in the heart, but not in prediabetic UCD-T2DM rats that express only nonamyloidogenic rat amylin and thus lack cardiac oligomeric amylin accumulation. Such an effect agrees well with previous data showing that the toxicity associated with amyloidogenic proteins is mediated by an initial increase in [Ca2+]i.20,25,37 Whereas the mechanisms underlying the increase of [Ca2+]i are not fully elucidated, our data suggest that an augmented passive trans-sarcolemmal Ca2+ flux is partly responsible. Amylin oligomers may also modulate the function of Ca2+ channels, as proposed in the pathology of Alzheimer disease.20,50 Elevated [Ca2+]i is involved in transcriptional regulation and hypertrophic signaling in the heart.40,41 Both CaMKII-HDAC and calciuneurin-NFAT hypertrophic pathways are activated in cardiac myocytes from prediabetic HIP rats (Figure 6A through 6D). Moreover, the calcineurin-NFAT pathway can be activated even by acute exposure of isolated myocytes to human amylin oligomers (Online Figure VIII). These data implicate the amylin oligomers as a trigger of hypertrophic and remodeling maladaptive changes in the heart (Figure 7). Prediabetic HIP rats show SERCA downregulation (Figure 6F), a common occurrence in diabetic cardiomyopathy,51 and increased level of the prohypertrophic hormone BNP (Figure 6E). SERCA downregulation causes further alterations in cardiac myocyte Ca2+ cycling by impairing Ca2+ transient relaxation, which leads to negative force-frequency relationship (Figure 5D) and elevated diastolic [Ca2+]i (Figure 5F). Slower Ca2+ transient relaxation and elevated diastolic [Ca2+]i may further activate the CaMKII-HDAC and calcineurin-NFAT transcriptional regulation/hypertrophic pathways and thus aggravate the cardiac hypertrophy and remodeling (Figure 7). Furthermore, impaired Ca2+ transient relaxation and elevated diastolic [Ca2+]i cause diastolic dysfunction in prediabetic HIP rats (Table). None of these alterations were present in prediabetic UCD-T2DM rats. Alterations in function and/or expression of proteins involved in cardiac Ca2+ cycling, including SERCA, and diastolic dysfunction have been reported in other rodent models of type 2 diabetes, but only after the onset of full-blown type 2 diabetes.7,38,39 Thus, our data indicate that cardiac amylin oligomer accumulation accelerates the occurrence of cardiac dysfunction and remodeling in diabetes.

Amylin Oligomer–Induced Cardiac Phenotype

At the whole-heart level, HIP rats show pathological signs of an infiltrative disease52 (Table). This is characterized by diastolic dysfunction (significantly reduced −dP/dtmin) and unchanged fractional shortening, which is one type of diastolic heart failure.52 Diastolic heart failure often progresses to systolic failure. Hemodynamics and echocardiographic measurements in HIP rats show indeed significantly reduced maximum rate of pressure fall (−dP/dtmin), an index of diastolic dysfunction, along with unchanged fractional shortening (Table). The left ventricular maximum systolic pressure is also significantly reduced in HIP rats (Table). Hence, prediabetic HIP rats display cardiac changes resembling the cardiac infiltrative disease in humans.52 Diastolic dysfunction is also important in the pathogenesis of diabetic cardiomyopathy.2–10,38,39 The molecular mechanisms underlying diastolic dysfunction in diabetic patients are poorly understood.2–10,38,39 Present data suggest that cardiac amylin oligomer accumulation is linked to Ca2+ dysregulation and pathological cardiac hypertrophy, which may accelerate the onset of diastolic dysfunction in diabetes.

The pathogenesis of diabetic cardiomyopathy is multifactorial and includes metabolic components2–10,38,39 that have not been studied here. Additional work to address any potential influences of the amylin oligomers on key metabolic processes in the heart is needed.

In conclusion, our data show that patients with obesity and type 2 diabetes accumulate amylin oligomers in the heart and suggest that this accelerates the development of cardiac dysfunction. We propose that detection and disruption of cardiac amylin buildup may be a predictor of myocardial dysfunction and a novel therapeutic target in diabetic cardiomyopathy.

Sources of Funding

This work was supported in part by the American Heart Association (BGIA2220165 to F.D.), National Science Foundation (CBET 1133339 to F.D.), National Institutes of Health (RO1-HL109501 to S.D.; RO1-HL089847, RO1-AG017022 to K.B.M.; RO1-HL077281, RO1-HL079071 to A.A.K.; HL075675, HL091333, AT003645, DK087307, HL107256 to P.J.H.; RO1-HL073162, RO1-HL061483 to H.T.; P01-HL080101 to D.M.B.), a Multicampus Award from the University of California, Office of the President (P.J.H.), and a Vision Grant from University of California-Davis Health System (F.D.).

Disclosures

None.

Acknowledgments

We thank Christine Malloy, RN, and James Graham, MSc, for technical help.

Footnotes

In December 2011, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.29 days.

Obesity and insulin resistance increase the risk for both type 2 diabetes and cardiac disease, but the underlying mechanisms remain poorly understood. In addition to hyperglycemia and dyslipidemia, patients with obesity and insulin resistance present also hyperinsulinemia and hyperamylinemia. Whereas the hyperinsulinemic response prevents a large fraction of insulin resistant patients from developing type 2 diabetes, the coincident hyperamylinemia leads to proteotoxicity and amyloid deposition in pancreatic islets. We show that amylin oligomers, fibrils and plaques also accumulate in failing hearts from obese and diabetic patients, but not in nonfailing hearts and failing hearts from lean, nondiabetic humans. Using rats transgenic for human amylin, we show that cardiac amylin oligomer accumulation causes myocyte Ca2+ dysregulation, activation of Ca2+-dependent pathological cardiac hypertrophy and remodeling, and diastolic dysfunction. Our data suggest that cardiac amylin accumulation accelerates the onset of diabetic cardiomyopathy. The present results show for the first time that amylin oligomers are a direct pathogenic link between pancreatic and cardiac disorders and an independent contributor to the multifactorial pathogenesis of diabetic cardiomyopathy. We propose that detection and disruption of cardiac amylin buildup may be a predictor of myocardial dysfunction and a novel therapeutic target in diabetic cardiomyopathy.